This invention relates generally to isotope dilution analysis and to chromatography, and has particular reference to a novel method for practicing isotope dilution analysis with the apparatus of chromatography.
In prior art isotope dilution analysis, an unknown quantity of analyte is determined through an isotopically labeled derivative of the analyte. In one embodiment of the art said derivative is radioactive. A known quantity of the radioisotope of known specific activity is added to the sample matrix, and a known quantity of the analyte-radioisotope mixture is isolated therefrom. The specific activity of the added radioisotope is decreased through dilution with the analyte, and the decrease is proportional to the quantity of analyte present. The specific activity determination requires a knowledge of the amount of radioactivity in the isolated material, and also a knowledge of the total mass of compound that contains the activity. Mass measurements are difficult to make in trace work, and isotope dilution analysis is not, therefore, generally applicable to trace analysis. To some extent the difficulty can be overcome by methods such as sub-stoichiometric analysis, but these methods need to be developed on a case-by-case basis.
In chromatography, an analyte mixture is injected into a fluid that flows through a column. The components of the mixture are separated on the column and the outflow from the column is monitored by a suitable detector. The analyte of interest is sensed as it elutes from the column.
The applicant is not aware of any prior art that has addressed itself to the practice of isotope dilution analysis through the apparatus of chromatography by exploiting differences in retention between analyte and isotope. The closest prior art known to the applicant is U.S. Pat. No. 3,929,410 issued to Schloss. This patent is directed to a method of indirect analysis where a saturated solution containing a known amount of radioactive standard is allowed to equilibrate with an unknown amount of the same compound in its unlabelled form, and quantitation of the unlabelled material is made on the basis of the amount of radioactive standard remaining in the saturated solution. The present invention differs from the Schloss patent in that it utilizes the apparatus of chromatography to effect the dilution of isotope. Also, temperature changes are not required in the present invention, and it is not necessary to precisely saturate a known amount of a liquid phase with a known amount of the radioactive standard.
Another related prior art method has been disclosed by Banerjee in U.S. Pat. No. 4,734,377. This patent utilizes a liquid chromatographic system with the eluent saturated with an indicator. The indicator solubilizes or displaces the analyte from the stationary phase. Analytes that cannot be detected directly can be indirectly visualized through the indicator. The present invention differs from the Banerjee patent in that detection is based on the isotope dilution principle, in that the indicator and analyte are chromatographically identical, and in that the analyte is selectively detected. Other prior art developed in the course of a preliminary search consists of Cortes and Stevens (J. Chromatogr. 295(1984)269-275), Parkin (J. Chromatogr. 303(1984)436-439), Banerjee (Anal. Chem. 57(1985) 2590-2592), Banerjee and Castrogivanni (J. Chromatogr. 396(1987) 169-175), Banerjee (Anal. Chem. 60(1988)1626-1629), and U.S. Pat. Nos. 3,666,854 and 3,768,979.